Development Of Transgenic Rice Lines With Resistance To Rice Black-Streaked Dwarf Virus Disease

Rice black-streaked dwarf virus(RBSDV)disease has been outbroken in many provinces in China in recent years.Once the rice plants were infected they could not be saved,causing severe yield losses.Due to lack of high resistance germplasm and major resistance gene to RBSDV disease,it is very difficult to develop resistant variety through traditional breeding method.In our lab,we have confirmed that overexpression of S6RNAi gene,which produces small RNA interference fragments specifically targeting RBSDV S6 gene,could be used to develop RBSDV high resistance variety.In this study,we used rice green tissue-specific promoter Osrbcs to drive S6RNAi expression and employed double T-DNA transformation system to develop marker-free and expression specific RBSDV resistance rice lines.The susceptible japonica rice variety Huaidao 5(HD5),widely planted in Jiangsu province,was used as transgenic receptor.The main results are as follows:1.We successfully generated a new double T-DNA plant expression construct'Osrbcs::S6RNAi' and transformed it into rice variety HD5 through Agrobacterium-mediated plant transformation method.We obtained 79 independent transgenic plants carrying S6RNAi gene,and around 97.4%of them carried marker gene(HPT)either.Through Southern blotting,we found that most of the transgenic plants carried more than two S6RNAi gene copies or HPT gene copies,and only few plants possessed single copy of foreign gene.Through PCR and Southern blotting detection on consecutive generations from each transgenic plant,we lastly obtained 5 marker-free transgenic lines.2.Through natural identification and artificial inoculation identification on some lines,we confirmed that Osrbcs::S6RNAi recombinants can be used to develop transgenic rice with high resistance to RBSDV.Among 79 independent transgenic plants,rice lines derived from independent transgenic plant '89'(hereafter named 89-derived lines)mostly showed high resistance to RBSDV.Under artificial inoculation condition,compared with control variety HD5 with more than 80%diseased plant,most lines derived from '89' only had less than 5%diseased plant,which also displayed high resistant performance in natural identification tests.Through Northern blotting,we found that most of 89-derived lines displayed strong expression of S6RNAi gene.Among 89-derived lines,we totally selected out 8 elite lines with high RBSDV resistance and 2 of them were marker-free lines.All the 8 lines contained 2 or 3 S6RNAi gene copies.Compared with the control variety HD5,most transgenic lines showed various alternation on agronomic traits,and 89-derived lines displayed well agronomic phenotype.3.We also analyzed the performance of some Ubi::S6RNAi transgenic lines,previously developed in our lab using constitutive promoter Ubi driving S6RNAi expression.In natural identification test,we obtained 1 or more marker-free lines from each of the 4 independent transformants(10-5,11-5,7-9,9-4)with single copy of S6RNAi gene.Some transformants were also used to cross with the receptor variety WLJ1 for further reducing inferior traits and recovering the WLJ1 background.From the progeny of each cross,we obtained a few lines(hereafter namely improved lines)with apparently improved agronomic performance.For instance,transformant 11-5 showed early senescence,while 11-5 derived improved lines all displayed normal phenotype without early senescence.Through Northern blotting,we confirmed that rice lines derived from the 4 independent transformants all showed strong S6RNA1 expression.By using inverse PCR technology,we determined that the location of Ubi::S6RNAi in transformants 10-5,11-5,7-9 and 9-4 are at 26.82 and 7.69 Mb of chromosome 6,and at 24.27 Mb and 4.97 Mb of chromosome 12,respectively.In total,this study provides valuable resources for further developing RBSDV resistant rice varieties,which is of great significance.